ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

sTREM-1 and TNF-α levels are associated with the clinical outcome of leprosy patients.

Leprosy reaction (LR) and physical disability (PD) are the most significant clinical complications of leprosy. Herein, we assessed the circulating serum-sTREM-1 and TNF-α levels and their genetic polymorphisms in leprosy. Serum-sTREM-1 and TNF-α levels were measured in leprosy patients (LP) before treatment (n = 51) and from their household contacts (HHCs; n = 25). DNA samples were genotyped using TREM-1 rs2234246 and TNF-α rs1800629-SNP in 210 LPs and 168 endemic controls. The circulating sTREM-1 and TNF-α levels are higher in the multibacillary form. The ROC curve of the serum-sTREM-1 levels was able to differentiate LR from non-LR and PD from non-PD. Similarly, LPs with serum-sTREM-1 levels >210 pg/ml have 3-fold and 6-fold higher chances of presenting with LR and PD, respectively. Genotypes CC+CT of the TREM-1 were associated with leprosy. Taken together, our analyses indicated that sTREM-1 and TNF-α play an important role in the pathogenesis of leprosy and provide promising biomarkers to assist in the diagnosis of leprosy complications.

A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Association between asthma, rhinitis and atopic dermatitis with leprosy: A case-control study.

Background Considering the cross-regulation of Th1 and Th2 responses, we hypothesised that atopic diseases (Th2) inhibit the protective Th1 immune response to Mycobacterium leprae and exacerbates leprosy. Objective In this study, we aimed to evaluate the association between leprosy and atopic diseases. Methods To evaluate the association of atopic diseases with leprosy, we conducted a case-control study that included leprosy patients (n = 333) and their household contacts (n = 93). The questionnaire from the International Study of Asthma and Allergies in Childhood, which is validated in several countries for epidemiological diagnosis of atopic diseases, was applied to determine the occurrence of atopic diseases, allergic rhinitis, asthma, and atopic dermatitis among leprosy patients and the household contacts. Results Considering clinical and epidemiological data, among the leprosy group 51.6% (n = 172) were determined to have at least one atopic disease, while atopy was observed less frequently at 40.86% among household contacts (n = 38). When two or more atopic diseases were assessed, the frequency was significantly higher among the leprosy patients than in the household contacts (21.9% vs. 11.8%; P-value = 0.03). Likewise, the frequency of asthma was significantly higher among leprosy patients (21%) than in the household contacts (10.8%; P-value = 0.02). Thus, our analyses revealed an association of atopic diseases with leprosy, with a significant linear increase in the occurrence of leprosy with an increase in the number of atopic diseases (P-value = 0.01). Limitation Due to the difficulties in recruiting household contacts that have prolonged contact with patients, but are not genetically related to the patient, the household contacts group is smaller than the leprosy patient group. Conclusion The data reveal an association between atopic diseases and leprosy outcomes. This knowledge could improve the treatment of leprosy patients with co-incident atopic diseases.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Leishmaniasis Vaccines: Applications of RNA Technology and Targeted Clinical Trial Designs.

Leishmania parasites cause a variety of discrete clinical diseases that present in regions where their specific sand fly vectors sustain transmission. Clinical and laboratory research indicate the potential of immunization to prevent leishmaniasis and a wide array of vaccine candidates have been proposed. Unfortunately, multiple factors have precluded advancement of more than a few Leishmania targeting vaccines to clinical trial. The recent maturation of RNA vaccines into licensed products in the context of COVID-19 indicates the likelihood of broader use of the technology. Herein, we discuss the potential benefits provided by RNA technology as an approach to address the bottlenecks encountered for Leishmania vaccines. Further, we outline a variety of strategies that could be used to more efficiently evaluate Leishmania vaccine efficacy, including controlled human infection models and initial use in a therapeutic setting, that could prioritize candidates before evaluation in larger, longer and more complicated field trials.

Gauging the skin resident Leishmania parasites through a loop mediated isothermal amplification (LAMP) assay in post-kala-azar dermal leishmaniasis.

Despite the availability of highly sensitive polymerase chain reaction (PCR)-based methods, the dearth of remotely deployable diagnostic tools circumvents the early and accurate detection of individuals with post-kala-azar dermal leishmaniasis (PKDL). Here, we evaluate a design-locked loop-mediated isothermal amplification (LAMP) assay to diagnose PKDL. A total of 76 snip-skin samples collected from individuals with probable PKDL (clinical presentation and a positive rK39 rapid diagnostic test (RDT)) were assessed by microscopy, qPCR, and LAMP. An equal number of age and sex-matched healthy controls were included to determine the specificity of the LAMP assay. The LAMP assay with a Qiagen DNA extraction (Q-LAMP) showed a promising sensitivity of 72.37% (95% CI: 60.91-82.01%) for identifying the PKDL cases. LAMP assay sensitivity declined when the DNA was extracted using a boil-spin method. Q-qPCR showed 68.42% (56.75-78.61%) sensitivity, comparable to LAMP and with an excellent agreement, whereas the microscopy exhibited a weak sensitivity of 39.47% (28.44-51.35%). When microscopy and/or qPCR were considered the gold standard, Q-LAMP exhibited an elevated sensitivity of 89.7% (95% CI: 78.83-96.11%) for detection of PKDL cases and Bayesian latent class modeling substantiated the excellent sensitivity of the assay. All healthy controls were found to be negative. Notwithstanding the optimum efficiency of the LAMP assay towards the detection of PKDL cases, further optimization of the boil-spin method is warranted to permit remote use of the assay.

Mycobacterium leprae induces Schwann cell proliferation and migration in a denervated milieu following intracutaneous excision axotomy in nine-banded armadillos.

Nine-banded armadillos develop peripheral neuropathy after experimental Mycobacterium leprae infection that recapitulates human disease. We used an intracutaneous excision axotomy model to assess the effect of infection duration by M. leprae on axonal sprouting and Schwan cell density. 34 armadillos (17 naïve and 17 M. leprae-infected) underwent 3 mm skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy 3 and 12-months post M. leprae inoculation. A traditional distal leg biopsy was obtained at 15mo for intraepidermal nerve fiber (IENF) density. Serial skin sections were immunostained against a axons (PGP9.5, GAP43), and Schwann cells (p75, s100) to visualize regenerating nerves. Regenerative axons and proliferation of Schwann cells was measured and the rate of growth at each time point was assessed. Increasing anti-PGL antibody titers and intraneural M. leprae confirmed infection. 15mo following infection, there was evidence of axon loss with reduced distal leg IENF versus naïve armadillos, p < 0.05. This was associated with an increase in Schwann cell density (11,062 ± 2905 vs. 7561 ± 2715 cells/mm3, p < 0.01). Following excisional biopsy epidermal reinnervation increased monotonically at 30, 60 and 90 days; the regeneration rate was highest at 30 days, and decreased at 60 and 90 days. The reinnervation rate was highest among animals infected for 3mo vs those infected for 12mo or naïve animals (mean ± SD, 27.8 ± 7.2 vs.16.2 ± 5.8vs. 15.3 ± 6.5 mm/mm3, p < 0.05). The infected armadillos displayed a sustained Schwann cell proliferation across axotomy time points and duration of infection (3mo:182 ± 26, 12mo: 256 ± 126, naive: 139 ± 49 cells/day, p < 0.05). M. leprae infection is associated with sustained Schwann cell proliferation and distal limb nerve fiber loss. Rates of epidermal reinnervation were highest 3mo after infection and normalized by 12 mo of infection. We postulate that excess Schwann cell proliferation is the main pathogenic process and is deleterious to sensory axons. There is a compensatory initial increase in regeneration rates that may be an attempt to compensate for the injury, but it is not sustained and eventually followed by axon loss. Aberrant Schwann cell proliferation may be a novel therapeutic target to interrupt the pathogenic cascade of M. leprae.

Evaluation of recombinase-based isothermal amplification assays for point-of-need detection of SARS-CoV-2 in resource-limited settings.

OBJECTIVES: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. METHODS: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. RESULTS: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18-85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58-92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40-9.76%). CONCLUSIONS: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.

The Importance of RNA-Based Vaccines in the Fight against COVID-19: An Overview.

In recent years, vaccine development using ribonucleic acid (RNA) has become the most promising and studied approach to produce safe and effective new vaccines, not only for prophylaxis but also as a treatment. The use of messenger RNA (mRNA) as an immunogenic has several advantages to vaccine development compared to other platforms, such as lower coast, the absence of cell cultures, and the possibility to combine different targets. During the COVID-19 pandemic, the use of mRNA as a vaccine became more relevant; two out of the four most widely applied vaccines against COVID-19 in the world are based on this platform. However, even though it presents advantages for vaccine application, mRNA technology faces several pivotal challenges to improve mRNA stability, delivery, and the potential to generate the related protein needed to induce a humoral- and T-cell-mediated immune response. The application of mRNA to vaccine development emerged as a powerful tool to fight against cancer and non-infectious and infectious diseases, for example, and represents a relevant research field for future decades. Based on these advantages, this review emphasizes mRNA and self-amplifying RNA (saRNA) for vaccine development, mainly to fight against COVID-19, together with the challenges related to this approach.

Novel polyprotein antigens designed for improved serodiagnosis of bovine tuberculosis.

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

Serum IgA Antibodies Specific to M. leprae Antigens as Biomarkers for Leprosy Detection and Household Contact Tracking.

Leprosy remains endemic in several developing countries, such as India and Brazil, in part due to delayed diagnosis that facilitates ongoing transmission. Although immunoglobulins against several Mycobacterium leprae antigens have been indicated for the early diagnosis, and IgA participates in the early stages of leprosy and in subclinical infection, relatively little research has examined anti-M. leprae IgA responses. Here, we investigated serum IgA reactivity against NDO-HSA, LID-1 and NDO-LID, in paucibacillary (PB) and multibacillary (MB) patients and their household contacts, using enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy of each ELISA was evaluated by receiver operating characteristic (ROC) curve analysis. Our data reveal elevated IgA serum levels against the three M. leprae specific antigens in MB patients, whereas IgA reactivity in PB patients was increased only to NDO-HSA. Further, MB and PB household contacts displayed higher IgA reactivity to NDO-HSA than non-endemic controls. Our data suggest measurement of serum IgA against NDO-HSA as an additional tool in the diagnosis and classification of the disease, with potential utility for household contact follow-up.

Evaluation of Loopamp™ Leishmania Detection Kit and Leishmania Antigen ELISA for Post-Elimination Detection and Management of Visceral Leishmaniasis in Bangladesh.

With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Leishmania Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the Leishmania antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS via silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the Leishmania antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the Leishmania antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.

Population-based, spatiotemporal modeling of social risk factors and mortality from schistosomiasis in Brazil between 1999 and 2018.

Schistosomiasis remains a significant public health concern in Brazil. To identify areas at, and social determinants of health (SDH) associated with, high-risk for schistosomiasis-related mortality from Brazil, we conducted a spatial and spatiotemporal modeling assessing all deaths confirmed in Brazil between 1999 and 2018. We used the segmented log-linear regression model to assess temporal trends, and the local empirical Bayesian estimator, the Global and Local Moran Index for spatial analysis. A total of 12,251 schistosomiasis-related deaths were reported in this period. Within the Mortality Information System (SIM) of the Brazilian Ministry of Health, the states of Alagoas (AL), Pernambuco (PE) and Sergipe (SE) recording the highest mortality rates: 2.21, 1.92 and 0.80 deaths/100,000 inhabitants, respectively. Analyses revealed an increase in the mean age of schistosomiasis-related deaths over the time assessed (APC = 0.9; p-value<0.05). Spatial analysis identified a concentration of municipalities presenting high risk of schistosomiasis-related mortality along the coastline of PE and AL. Similarly, we identified the formation of high space-time clusters in municipalities in the states of PE, AL, SE, Bahia, and Minas Gerais. Finally, mortality rates showed a significant correlation with 96.96% of SDH indices. The data reveal additional important changes in schistosomiasis-related deaths in Brazil between 1999 and 2018, such as a slow reduction among males (unlike females that displayed no change). Regardless, our analyses indicates that schistosomiasis continues to have the greatest detrimental impact in poor regions of Brazil and suggest the need for enhancement of current control measures to accelerate progress.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Skin tests for the detection of Mycobacterial infections: achievements, current perspectives, and implications for other diseases.

Immunological and molecular advances have modernized diagnostic testing for many diseases. Although interferon gamma-release and polymerase chain reaction assays have been developed to detect Mycobacterium tuberculosis (Mtb) infection, purified protein derivative (PPD)-based tuberculin skin testing (TST) remains the most widely used method. Indeed, the TST is a simple and cost-effective tool that can be easily applied for widespread screening for Mtb infection. However, the lack of specificity has been a limitation of these tests, and, more recently, supply issues have arisen. Building upon the skin tests that historically have been used within TB and leprosy control programs, we discuss recent developments using modern technologies for improving mycobacterial skin testing as well as practical advantages inherent to the technique. Furthermore, we outline how this knowledge could be applied to develop similar tests that could benefit diagnostic strategies for other infections. KEY POINTS: • Skin testing provides a significantly cheaper alternative to most modern technologies. • Skin tests provide a lab-independent diagnostic strategy that can be widely administered. • Diseases for which T cell responses are more robust or durable than antibody responses are accessible for skin testing.

Evolution of antigen-specific immune responses in cutaneous leishmaniasis patients.

AIMS: Despite immunization appearing to be the most appropriate strategy for long-term control of the vector-borne leishmaniases, no sustainable vaccine is currently available against any form of leishmaniasis. We therefore evaluated, in the context of vaccine antigen candidates, antigen-specific immune response at various stages of cutaneous leishmaniasis (CL). METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers and CL patients (caused by either Leishmania major or L tropica) were incubated with crude Leishmania proteins (soluble Leishmania antigen; SLA), single recombinant proteins (TSA, LeIF, LmSTI1) or chimeric fusion proteins (LEISH-F2 and LEISH-F3). The concentrations of immune modulatory cytokines were then determined. While we did not detect appreciable antigen-specific IL-5 secretion, SLA induced secretion of interleukin (IL)-10 in cultures from early active lesion CL patients and even from healthy individuals. Conversely, interferon (IFN)-γ responses to SLA and recombinant proteins followed a similar pattern, developing only in the late active CL lesion phase. Once established, antigen-specific IFN-γ responses persisted in cured CL patients. CONCLUSION: Together, our results provide further insight into the development of immune responses during CL and further validate the selection of LEISH-F2 and LEISH-F3 as vaccine antigen candidates.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.